With use of multiple- and single-colony expansion procedures, the results of susceptibility testing of Helicobacter pylori isolates from patients with duodenal ulcer were assessed by Etest. The H. pylori genotype was assessed by repetitive extragenic palindrome-based polymerase chain reaction (REP-PCR). There was a high degree of genotypic heterogeneity between different patients, but a single REP-PCR pattern was found for 92% of patients. In contrast, a high degree of phenotypic heterogeneity was shown among the isolated colonies. Antibiogram susceptibility patterns differed only with respect to metronidazole but not with respect to clarithromycin or amoxicillin. The 42% rate of resistance to metronidazole determined with use of the conventional multiple-strains expansion method was increased to 92% when the single-colony expansion method was used. Similarly, dual clarithromycin/metronidazole resistance was increased from 8% to 42% with single-colony expansion. Despite evidence of a single genotype in most patients, single-colony expansion shows that routine susceptibility testing may greatly underestimate the frequency of metronidazole resistance.